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The Sinorhizobium meliloti ExoS/ChvI two component regulatory system is required for N2-fixing symbiosis and exopolysaccharide synthesis. Orthologous systems are present in other Alphaproteobacteria, and in many instances have been shown to be necessary for normal interactions with corresponding eukaryotic hosts. Only a few transcriptional regulation targets have been determined, and as a result there is limited understanding of the mechanisms that are controlled by the system.
In an attempt to better define the members of the regulon, we have applied a simple in vitro electrophoretic screen for DNA fragments that are bound by the ChvI response regulator protein. Several putative transcriptional targets were identified and three were further examined by reporter gene fusion experiments for transcriptional regulation. Two were confirmed to be repressed by ChvI, while one was activated by ChvI.
Our results suggest a role for ChvI as both a direct activator and repressor of transcription. The identities and functions of many of these genes suggest explanations for some aspects of the pleiotropic phenotype of exoS and chvI mutants. This work paves the way for in depth characterization of the ExoS/ChvI regulon and its potential role in directing bacteria-host relationships.
Keywords:Rhizobia; Response regulator; DNA-binding; Exopolysaccharide; Alfalfa nodulation; chvI regulon; Signal transduction; Transcriptional regulation; DNA binding assay
Two-component regulatory systems (TCRS) are the most abundant and widespread transcriptional regulators in bacteria, as indicated by the number of instances of the Pfam PF00072 response regulator receiver domain [1]. Bacterial genomes typically contain dozens to hundreds of these systems [2]. Response regulator domains of transcriptional regulatory proteins are phosphorylated by cognate sensor histidine kinase proteins in response to changes in environment or growth conditions [3]. This phosphorylation results in conformational change of the response regulator protein, leading to transcriptional activation or repression. Even with the recognized importance of these systems, very few of them have been characterized with regard to the signal input and the regulatory targets.
The ExoS/ChvI two-component regulatory system, consisting of the membrane-spanning histidine protein kinase ExoS and the response regulator ChvI, is found in alphaproteobacterial genomes. In Agrobacterium tumefaciens, the ChvG/ChvI system is vital for plant tumor formation, and mutants are sensitive to acidic pH and detergents [4]. The BvrS/BvrR system of Brucella abortus is required for virulence [5] and has a broad impact on cell envelope as well as carbon and nitrogen metabolism [6]. The Bartonella henselae BatR/BatS system is also involved in regulating virulence-associated genes [7]. Analysis of a mutant of the ExoS homolog of Rhizobium leguminosarum confirmed its requirement for successful nodule invasion and nitrogen fixation [8]. This mutant also had a destabilized outer membrane, associated with reduction of ropB expression, as well as increased accumulation of intracellular poly-3-hydroxybutrate (PHB), and reduction in exopolysaccharide production. In all cases studied, ExoS/ChvI TCRS and its orthologs play a role, although not well understood, in the bacterial-host interaction.
Sinorhizobium meliloti exoS was first identified through a Tn5 insertion mutant that resulted in overproduction of exopolysaccharide due to disruption of the membrane-spanning portion of the protein, causing constitutive activation of the kinase activity, thus resulting in constant phosphorylation of ChvI [9]. Null mutants of exoS and chvI are able to trigger the formation of nodules, but those nodules do not develop normally and they do not fix nitrogen [10]. The mutants do not grow on complex or in liquid media, and cultivation on defined agar-media is challenging, a condition that prompted an early conclusion that exoS and chvI are essential for S. meliloti viability [11]. A chvI deletion mutant demonstrated enhanced motility, and reduction in PHB accumulation, the opposite of what was found for a R. leguminosarum exoS homolog mutant [12]. Similar to the R. leguminosarum mutant [8], alterations in LPS were observed. Also isolated in the Tn5 screen that yielded the constitutively activated exopolysaccharide overproducing exoS mutant was a mutant of exoR[9]. Evidence has been provided to suggest a direct interaction of ExoR with ExoS in the periplasm, with ExoR binding contributing to the maintenance of ExoS in an inactive conformation [13]. Furthermore, it has been proposed that cleavage of ExoR is induced by some yet unknown environmental signal during infection of the host plant, and this might modulate its ability to bind ExoS [14], resulting in its activation and regulation of the target genes.
The exoS gene is situated within an operon along with hprK, part of an incomplete phosphotransferase system (PTS) in Alphaproteobacteria. In S. meliloti, HprK is involved in succinate mediated catabolite repression [15]. The establishment of a direct functional or regulatory link between the incomplete PTS and the ExoS/ChvI TCRS has been elusive, partly because the systems have often been studied in isolation.
Given the pleotropic nature of the exoS and chvI null mutants [10], investigation of gene expression using transcriptomics and proteomics might prove less than satisfactory, as the expression of many genes that are not direct regulatory targets is likely to be altered due to physiological changes in the cell. Indeed transcriptomics have identified hundreds of genes whose expression is affected by the exoS96::Tn5 mutation [16]. Comparison of transcriptomes from two different chvI mutant strains (gain-of-function versus reduced-function) narrowed the set of genes regulated by ChvI and subsequently facilitated the identification by gel shift assays of three intergenic regions binding ChvI [17] and the determination of an 11-bp-long putative ChvI binding motif. However, for the majority of genes identified as being differentially expressed in a ChvI dependent manner in that study, including the succinoglycan synthesis genes, no binding to upstream regions could be demonstrated. As an alternative, we applied a method to screen for DNA fragments that were directly bound by the ChvI transcriptional regulator. Analysis of these targets suggests important metabolic pathways affected by ChvI regulation. In return, these new findings directed us to uncover better conditions for cultivation of the loss-of-function chvI mutants. Further analyses with reporter gene fusion assays confirmed the direct role of ChvI as a repressor for the rhizobactin and SMc00261 operons. It also confirmed the previously discovered direct activation of the msbA2 operon by ChvI. Methods developed here to identify ChvI targets have proved to be efficient and could be applied to other response regulators.
Application of electrophoretic mobility shift assay to the identification of ChvI-regulated genes
To better understand the role of ChvI as a response regulator, it is necessary to identify genes whose transcription is directly influenced by ChvI. To identify specific DNA sequences from plasmid or genomic DNA for which ChvI might have binding affinity, we adapted a method using the electrophoretic mobility shift principle [18]. The plasmid DNA electrophoretic mobility shift assay (PD.EMSA) and genomic DNA electrophoretic mobility shift assay (GD.EMSA) methods involve incubation of purified DNA-binding protein with fractionated DNA, followed by electrophoresis through a native polyacrylamide gel using sodium boric acid (SB) buffer. In this study, the restriction endonuclease Bsp143I was used for DNA fragmentation. The use of SB buffer, a low conductivity medium, and a 14-cm gel as well as running the gel for 3–6 hours at low voltage, allowed unbound DNA fragments to migrate far from the top of the gel while ChvI-bound fragments remained near the wells (see Additional file 1). Inclusion of EDTA in the buffer resulted in no retardation of electrophoretic mobility suggesting an involvement of the putative Mg2+ site for ChvI-DNA interaction (see Additional file 2). The slower migrating bands were excised from the gel, purified, and cloned into pUC18 vector from which the insert DNA could be sequenced from each end to determine the extent of each fragment.
Additional file 1. Gel image of PD.EMSA to compare DNA shifts on 6-cm versus 14-cm 5% nondenaturing polyacrylamide gel and using SB buffer. Prior to the electrophoresis, the Bsp143I restricted pTC198 plasmid was incubated or not with the HisTag-ChvI protein.
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Additional file 2. Gel image of PD.EMSA to compare ChvI binding specificity in presence of EDTA or acetylphosphate. A 5% nondenaturing polyacrylamide gel made with TB buffer was used for the electrophoresis of the EcoRI-PstI double restricted pLB102 plasmid. The plasmid DNA was incubated or not with HisTag-ChvI protein in presence or not of EDTA and in presence or not of acetylphosphate (AP) prior to the electrophoresis.
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Bsp143I-digested pTC198 plasmid DNA was used to perform PD.EMSA (see Additional file 1). This pUC19 clone contains a 5-kb KpnI-fragment from S. meliloti Rm1021 spanning across the entire chvI-hprK genomic sequence including the intergenic region between pckA and chvI[10]. This plasmid was employed to optimize the method with a smaller number of fragments than with genomic DNA, thus providing a better resolution on the gel but also increasing the chances of binding to areas surrounding chvI and exoS to test for possible autoregulation of ExoS/ChvI. Regulation of the adjacent gene pckA by chvG-chvI has been previously shown for A. tumefaciens using reporter gene fusion assays [19], therefore this experiment was also aimed at testing if S. meliloti ChvI could bind upstream of pckA.
Following the excision of electrophoretic bands from PD.EMSA of pTC198, DNA fragments were cloned into BamHI-linearized pUC18 and sequenced from both ends. Out of four inserts sequenced, three represent a 176-bp fragment (genomic origin from 48523 to 48699) coding for the region upstream of SMc02753, including its start codon. A single clone contained a 395-bp region spanning the upstream sequence of chvI and past the translational start site (genomic origin from 51887 to 52281). These results suggest that ChvI might autoregulate its transcription but most importantly, it shows a direct binding affinity between the ChvI and the upstream sequence of manXhpr operon part of the PTS system. The ChvI binding to the 176-bp fragment was also confirmed by performing a gel shift assay using a PCR-amplified DNA fragment from pLB102 and the purified ChvI protein (data not shown). Further delineation of this binding was not performed.
After GD.EMSA, the examination of 27 clones resulted in the identification of a large number of additional potential targets for ChvI regulation (Table 1). Fragments ranged in size from 67 bp to 595 bp. Interestingly, the majority of fragments identified were found to be in predicted coding sequences rather than in intergenic regions. Moreover, ChvI-binding fragments are widely distributed across the genome and are not confined to a particular metabolic pathway. Although no one fragment was identified more than once, two non-contiguous fragments that are part of the same gene (rhtX) were independently cloned and sequenced. Fragments from the exoS-chvI region were not among the sequenced clones. Reporter gene fusions were used to confirm the chvI-regulated transcription of three selected genes (see below). These genes were selected based on their availability from a random fusion library [20], to test a mix of inter and intra fragments and to validate the previously described regulation of the msbA2 gene cluster.
Table 1. DNA fragments recovered from GD.EMSA and genes potentially regulated by ChvI
Potential functions of identified ChvI-regulated genes
Genes potentially regulated by ChvI are of diverse function (Table 1). Because DNA fragments binding ChvI are often found within a coding sequence and not in intergenic areas, it is difficult to predict if ChvI acts as an activator of an adjacent gene or a repressor of the gene it binds within. In several cases, such as the rhizobactin gene cluster and the msbA2 gene cluster, the ChvI-binding fragment is found in the first gene of what is predicted to be an operon. Table 1 lists genes found closest to a ChvI-binding DNA fragment but it is possible in many instances that genes further downstream could be part of the same transcript and also be ChvI-regulated. It is also important to note that the sequenced fragments are a subset of cloned fragments and other ChvI targets likely exist. Using the list of potentially ChvI-regulated genes obtained, we queried databases for functional relationships between targets: MetaCyc [21], KEGG [22] and STRING 8.1 [23]. Based on these analyses, a number of functional linkages may be made between some potential ChvI targets.
Two fragments (F15 and F6) are linked to lactose catabolism. One is found in front of the lacFGZ1K gene cluster and the second is found in SMc00589 (a conserved hypothetical protein), about 300 bp upstream of gal (Smc00588). The lacFGZ1K gene cluster encodes genes for lactose ABC-transporter and a β-galactosidase (E.C. 22.214.171.124). β-D-galactose is degraded through the De Ley-Doudoroff pathway in S. meliloti[24,25] and gal codes for the galactose dehydrogenase (EC 18.104.22.168) of this pathway.
Two other fragments (F7 and F5) suggest that ChvI is involved in regulating phospholipid biosynthesis. One fragment is found in SMc02076 (cls) and another one is found in SMc00550, about 300 bp upstream of psd (SMc00551) and followed by pssA (SMc00552). Cardiolipin is produced in S. meliloti and the only gene coding for a cardiolipin synthetase is cls[26]. Interestingly, this gene is located about 1 kb downstream of the exoS-associated gene exoR. Proteins encoded by psd (phosphatidylcholine decarboxylase) and pssA (phosphatidylserine synthase) are responsible for the biosynthesis of phosphatidylethanolamine and phosphatidylserine respectively, and both of these phospholipids are also intermediates for phosphatidylcholine biosynthesis [27]. Mutants of these genes exhibit deficiencies in alfalfa symbiosis [27]. Aside from phospholipids synthesis, another link was found between SMc00550 and msbA2 using STRING 8.1. These two genes are homologs and might have similar functions. The fragment F8 found in SMc00262, a putative 3-ketoacyl-CoA thiolase, followed by SMc00261, a putative fatty-acid-CoA ligase, also suggests regulation of lipid metabolism. These genes are putatively involved in fatty acid β-oxidation.
ChvI was also found to bind fragments from genes involved in peptide and methionine transport. A fragment (F13) belongs to the upstream sequence of SMc03267 and four genes encoding a putative dipeptidase and a putative dipeptide ABC-type transporter. Another fragment (F19) is from SMb20478, part of a gene cluster coding for another dipeptide ABC-transporter. MetN involved in importing methionine also has a fragment of its gene having affinity for ChvI.
A fragment found in thiC (F23) and another found in hisB (F1) do not present a directly evident link between the thiamine and histidine biosynthesis pathways they are respectively involved in but there is an indirect metabolic link that can be followed in MetaCyc, KEGG and in STRING. ThiC catalyzes the reaction between 5-aminoimidazole ribonucleotide (AIR) and hydroxymethylpyrimidine phosphate (HMP-P) in the thiamine biosynthesis pathway (Figure 1). AIR is biosynthesized from 5-phosphoribosyl 1-pyrophosphate (PRPP). PRPP is also required for the synthesis of histidine. In STRING this link is made through pur genes, which code for enzymes involved in purine synthesis. Pyrimidine, purine and pyridine nucleotide synthesis pathways are all dependent on the availability of PRPP.
Figure 1. 5-Phosphoribosyl 1-pyrophosphate (PRPP) metabolic pathway and the potential role of ChvI in regulating downstream biosynthesis pathways. Grey boxes represent genes potentially regulated by ChvI. Uridine-5'-phosphate (UMP), uridine-5'-diphosphate (UDP), uridine-5'-triphosphate (UTP), hydroxymethylpyrimidine phosphate (HMP-P), 4-amino-5-hydroxymethyl-2-methylpyrimidine-pyrophosphate (HMP-PP), 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (THZ-P), 5-phospho-β-D-ribosyl-amine (PRA), 5-phospho-ribosyl-glycineamide (GAR), 5'-phosphoribosyl-N-formylglycineamide (FGAR), 5-phosphoribosyl-N-formylglycineamidine (FGAM), 5-aminoimidazole ribonucleotide (AIR), 4-carboxyaminoimidazole ribonucleotide (CAIR), 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole (SAICAR), aminoimidazole carboxamide ribonucleotide (AICAR), phosphoribosyl-formamido-carboxamide (FAICAR), inosine-5'-phosphate (IMP), phosphoribosyl-ATP (PR-ATP), phosphoribosyl-AMP (PR-AMP), phosphoribosylformiminoAICAR-P (PRoFAR), phosphoribulosylformimino-AICAR-P (PRFAR), D-erythro-imidazole-glycerol-phosphate (IGP).
Following these analyses, we could not find a direct link between these potentially ChvI-regulated genes and the exopolysaccharide biosynthesis pathways, central to one of the most important phenotypes of the chvI mutant strain [10]. This is absolutely consistent with other experimental work that has failed to find direct binding of ChvI to exopolysaccharide synthesis gene upstream regions [17]. However, an indirect link is suggested from the regulation of thiamine and histidine biosynthesis (Figure 1). These pathways are inter-related with the synthesis of pyrimidine and consequently the availability of UTP required for the synthesis of UDP-glucose. Perhaps the imbalance caused by deregulating thiamine and histidine synthesis affects UDP-glucose synthesis and therefore polysaccharide production. To test this hypothesis, we added 0.1% uracil to the MM9-succinate minimal media and this improved significantly the growth of the chvI mutant strain, although still not to a level comparable to the wild-type (Table 2). However, an important finding from these experiments is that the addition of uracil allows the chvI null mutant strain to grow in liquid media. From carbon source utilization analyses performed in a previous work [10], proline or ornithine are good carbon sources for the chvI mutant strains, therefore 0.1% proline was added to MM9-succinate media supplemented also with 0.1% uracil. This improved the growth of the mutant strain even further (Table 2).
Table 2. Growth rate constants of chvI261 mutant strain grown in MM9-succinate liquid media and with the addition of uracil and/or proline to the growth media
Confirmation of ChvI involvement in transcriptional regulation of identified target genes
Having identified genes that might be regulated by ChvI and conditions allowing the growth of the chvI mutant strain in liquid media, we used strains from a S. meliloti fusion library [20] to confirm the regulation at transcriptional levels. The library had been constructed using a vector that forms gene fusions to the reporter genes gfp+/lacZ or gusA/tdimer2(12) depending on the orientation of the insert. Because of the possible involvement of ChvI in regulating the S. meliloti lac operon, we selected gusA fusion strains to measure transcriptional activity using the β-glucuronidase assay. Gene fusions were transduced into chvI mutant SmUW38 and into the wild-type strain Rm1021, and then assayed for β-glucuronidase activity and compared. These assays have been applied to three operons identified by the DNA binding assays, confirming the regulation of all three operons by ChvI, and also demonstrating that ChvI can function as either an activator or a repressor, depending on the target gene. The transcription assay with a housekeeping gene in the two genetic backgrounds (wild-type versus chvI261) was not tested. However, we did examine expression of the gene SMa2295 with a fusion upstream of the ChvI binding site and the results showed low and not significant GusA activity difference between the two genotype backgrounds (23 versus 30 Miller Units).
ChvI-bound fragment F20 was identified within SMb21188, the first gene of a predicted four-gene operon, and therefore we tested three gene fusions to SMb21189, SMb21190, and msbA2 (SMb21191) (Figure 2B). These fusions had a much higher expression level in the wild-type than in chvI mutant background (Figure 2A). These results suggest that ChvI is responsible for activation of the co-transcription of SMb21189, SMb21190, and msbA2 genes. Using a neural network promoter prediction tool [28], we predicted a putative transcriptional start site (P2) adjacent to the area containing a ChvI binding site (B). Another putative transcriptional start site (P1) further upstream from SMb21188 suggests that transcription might be directed from two differently regulated promoters, only one of which would include the SMb21188 gene.
Figure 2. Transcriptional fusion assays and the msbA2 operon. (A) GusA activities were measured for fusions in genes SMb21189, SMb21190, and msbA2 in wild-type (Rm1021) and chvI261 mutant (SmUW38) strain backgrounds. No GusA activities above background levels were detected for fusions to SMb21189 and SMb21190 in the chvI261 mutant strain background. (B) In the operon diagram, F1, F2, and F3 represent the positions of the fusions to SMb21189, SMb21190 and msbA2 respectively. The grey box (B) represents the region for ChvI binding, and P1 and P2 are predicted promoters.
Reporter gene fusion assays and promoter prediction were done with fusions in genes SMc00262 and SMc00261, which are predicted to encode a 3-ketoacyl-CoA thiolase and a fatty-acid-CoA ligase respectively (Figure 3B). In this case, a promoter was predicted immediately upstream of the ChvI binding area in SMc00262 and accordingly the fusions further downstream in SMc00262 and in SMc00261 presented higher expression levels in chvI mutant strains than in wild type (Figure 3A). These results suggest that ChvI acts by repressing the transcription of the SMc00264 – SMc00259 operon.
Figure 3. Transcriptional fusion assays and the SMc00261 operon. (A) GusA activities were measured for fusions in genes SMc00262 and SMc00261 in wild-type (Rm1021) and chvI261 mutant (SmUW38) strain backgrounds. (B) In the operon diagram, F1 and F2 represent the position of the fusions to SMc00262 and SMc00261 respectively. The grey box (B) represents the region for ChvI binding, and P1, P2 and P3 are predicted promoters.
S. meliloti produces an iron-siderophore, rhizobactin 1021, under iron-depleted conditions [29]. Genes for the synthesis and transport of rhizobactin are clustered in an operon [30]. The rhizobactin transporter coding sequence (rhtX, SMa2337) was found to contain two DNA fragments binding ChvI (Table 1 and Figure 4B). We tested a fusion following the first binding site (B1) and two other fusions further in rhbB (SMa2402; diaminobutyrate decarboxylase, EC 22.214.171.124) and in rhbF (SMa2410). The promoter prediction suggests the presence of a promoter before rhtX and another one before rhbA. The β-glucuronidase assays presented a higher expression in chvI background for all three fusions. This suggests that ChvI represses the expression of genes required for the synthesis and transport of rhizobactin 1021. Both binding areas seem to be important in repressing the transcription as shown by a higher expression in the fusion found before the second binding.
Figure 4. Transcriptional fusion assays and the rhizobactin operon. (A) GusA activities were measured for fusions in genes rhtX, rhbB and rhbF in wild-type (Rm1021) and chvI261 mutant (SmUW38) strain backgrounds. (B) The rhizobactin genes are clustered in one operon, F1 F2 and F3 represent the positions of the fusions to rhtX, rhtB, and rhbF respectively. The grey boxes (B1 and B2) represent the possible position for ChvI binding, and P1 and P2 are predicted promoters.
The high basal level of the negatively regulated operons is not really unexpected given that we do not know the repressing conditions, and also the likelihood of multiple regulatory systems acting on these genes. These experiments involved the comparison of gene expression in genetic backgrounds that resulted in differences only in the presence / absence of the ChvI regulator. Otherwise, the environmental conditions were not altered.
An adaptation of methods to perform gel electrophoresis mobility shift assays allowed us to identify DNA fragments with higher affinity for ChvI. Analyses of these results force us to revise our earlier perceptions following phenotypic analyses of ExoS/ChvI as mainly a regulatory system for exopolysaccharide production. Our results suggest that the ChvI regulon includes genes from diverse pathways. Moreover, ChvI appears to have a dual regulatory role, activating and repressing different operons. The total number of targets likely far outnumbers the 27 fragments that we pulled out in our screen, especially considering that we did not hit the same fragment more than once, and we also did not find a few other targets that had previously been shown to be bound by ChvI.
The approach used in our study is highly complementary to the microarray and directed DNA binding study of Chen et al. [17] that resulted in the identification of several potential regulatory targets of ExoS/ChvI and the prediction of a consensus binding sequence. It is important to note, however, that of 19 upstream regions tested, binding was only detected to three (ropB1, SMb21440, SMc01580), and a putative consensus sequence was determined using some upstream regions to which binding had not been demonstrated. Confirmation of this consensus binding sequence awaits more detailed DNA footprinting experiments on a larger number of identified targets. It is possible that many ChvI-repressed genes may not have been detected in that study due to the use of a constitutively activated variant of the ChvI protein that might not have been able to function as a repressor.
The binding of ChvI within SMa2337 (rhtX) to repress rhtXrhbABCDEF gene transcription could suggest that following the sensing of a signal other than the presence of iron, ExoS/ChvI represses genes for rhizobactin 1021 production. This operon is known to be upregulated by RhrA in iron-depleted conditions [31] and downregulated by RirA in iron-replete conditions [32]. Our fusion assay results confirmed that the rhizobactin operon is highly expressed in M9-minimal media (iron-depleted conditions). However, this expression is even higher in strains with the chvI null mutation. Iron is an important micronutrient found in many cofactors required for cytochrome and nitrogenase activity. Its acquisition however is difficult for two main reasons. First, it is poorly soluble at pH 7, and secondly, a high concentration of iron can cause the generation of hydroxy radicals. Bacteria produce siderophores to scavenge iron and therefore control iron availability. A tight control over the production of siderophore is thus important. The lack or the overproduction of rhizobactin 1021 by S. meliloti impairs the symbiotic relationship with alfalfa [29]. Mutation of rirA derepresses rhizobactin production and as a result causes a growth defect of the strain relative to the presence of iron [33]. The reduced viability of the rirA mutant due to oxidative stress suggested that perhaps this strain would also be affected in its symbiotic properties but it was not the case [33]. This study suggested that in planta another unknown regulatory system might control the production of rhizobactin. Whether ExoS/ChvI might be the system responsible awaits further investigation.
Another important finding is the confirmation that ChvI is involved in activation of the expression of SMb21189, SMb21190, and msbA2. These genes have only been described recently in the literature although msbA2 in particular may play an important but incompletely defined role in symbiosis [34,35], and the operon has already been shown to be subject to ChvI regulation [17]. SMb21189 and SMb21190 encode glycosyltransferases and msbA2 is part of an ABC-transporter family involved in macromolecule export. The above mentioned recent studies proposed that the operon including SMb21188, a putative acyltransferase, is involved in the production and export of an unknown polysaccharide which uses intermediates from the succinoglycan production pathway. The regulation of this operon by ExoS/ChvI is therefore the closest link to the succinoglycan-deficient phenotype of exoS and chvI mutant strains. Although this ChvI-regulated operon is not required for succinoglycan production it seems to be functionally related to succinoglycan production.
The third operon that we confirmed to be differentially regulated by ChvI encodes proteins putatively involved in fatty acid β-oxidation. SMc00262 putatively produces a 3-ketoacyl-CoA and SMc00261, a fatty-acid-CoA ligase. These genes are also followed by SMc00260 coding for a putative short-chain dehydrogenase and SMc00259 coding for a hypothetical protein. Upstream of these genes lay genes for a transcriptional regulator of the IclR family (SMc00263) and another short-chain dehydrogenase (SMc00264). Our earlier studies failed to demonstrate a phenotype for SMc00260 and SMc00264 mutants [36]. A tripartite ATP-independent periplasmic (TRAP) transporter system upregulated by the presence of acetoacetate and 3-methyl oxovaleric acid is encoded by genes in the same orientation upstream of SMc00264 [37]. All these genes are organized in the same orientation and close enough to each other to be part of the same transcript. However, our finding of a ChvI binding site in SMc00262, after the gene encoding the IclR regulator, suggests a complex regulation of these genes. In fact, a N-Acyl homoserine lactone (AHL) also impacts on their expression [38]. The fatty-acid-CoA ligase (SMc00261) has been found differentially accumulated in early log phase cultures of S. meliloti Rm1021 treated for 2 hours with 3-oxo-C16:1-HL while the periplasmic binding protein (SMc00265) accumulated in stationary phase cultures independently of the presence of AHLs. Perhaps under conditions that activate ChvI, the first part of the gene cluster is upregulated to allow the import of an organic acid but the second part responsible for its degradation and entry in the TCA cycle is downregulated. This hypothesis would suggest the use of this organic acid, under certain conditions, as a readily available building block rather than an energy source.
An important finding from this work is that uracil and proline improved the growth of the chvI mutant. This finding now allows us to culture the mutant strain in liquid media, greatly facilitating experimental analysis. Binding of ChvI in thiC (SMb20615) and in hisB (SMc02574), perhaps to repress the thiamine and histidine biosynthesis operons, made us hypothesize that a derepression of these operons in exoS or chvI mutants could lead to a deficiency in UTP formation and could explain the pleiotropy of these mutants. Rhizobial purine and pyrimidine auxotrophic mutants have been found affected in polysaccharides synthesis and plant invasion [39-42]. Further work needs to be done to confirm that chvI mutant auxotrophy is truly caused by a derepression of operons for thiamine and histidine biosynthesis.
We have identified a number of putative direct targets of ChvI, many of which are consistent with the pleotropic phenotype of exoS and chvI mutants. We also demonstrated that ChvI may act as a repressor or activator of gene expression, and surprisingly ChvI seems to often bind within predicted protein coding sequences. The bias is often to only consider intergenic regions for locations of potential regulatory sites. However, we note that the Fur regulator of Helicobacter pylori is just one example of a transcriptional regulatory protein that has targets within polycistronic operons and acts as a repressor and an activator of gene expression [43]. The tendency to search for transcriptional cis-regulatory elements in intergenic areas rather than considering equally regions internal to ORFs may need to be revisited. GD.EMSA or Chromatin-Immunoprecipitation (ChIP) techniques are examples of techniques that do not have a bias towards intergenic or intragenic areas and their usage certainly make important contributions to our knowledge about transcriptional regulation.
Although this study has uncovered new facets of the ExoS/ChvI regulation, the regulatory signal is still unknown. However, a number of new hypotheses emerge. Several genes identified in this study as possibly ChvI-regulated are involved in amino acid and peptide metabolism and transport. In Rhizobium leguminosarum bv. viciae VF39SM, peptides have been shown to increase the expression of the outer membrane protein ropB in a ChvG-dependent manner [44]. Perhaps ExoS and/or ExoR sense(s) peptides. Also, our work suggests a direct regulatory link between the PTS and the ExoS/ChvI systems; do these systems partner to coordinate the C and N metabolism as suggested by recent work in B. melitensis[45]? With several putative ChvI-targeted genes now identified, tools necessary to test these hypotheses are available. We are cognizant, however, of the fact that our screen was not saturating, and we will endeavor to adapt the method for higher throughput so that we have a better understanding of the complete ChvI regulon and the nature of the ChvI consensus binding sequence.
Bacterial strains, plasmids and growth conditions
Strains and plasmids used in this study are listed in Table 3. Growth conditions are as described previously [10] or as described in subsequent procedures.
Table 3. Bacterial strains, plasmids and primers used in this study
Cloning of chvI for His•Tag-ChvI expression and purification
S. meliloti Rm1021 chvI was PCR amplified using primers LB5 and LB6 (Table 3). The 800-bp PCR fragment was gel-purified and then cloned in pGEM®-T Easy vector. Plasmid pLB010 with the insert in the correct orientation for expression was verified by DNA sequence analysis. NotI chvI-containing fragment was then cut out of pLB010 and ligated to NotI-digested pET-30a, generating a N-terminal His•Tag fusion pJF011. E. coli BL21(DE3)pLysS clones carrying the pJF011 plasmid were confirmed for His•Tag-ChvI production by western blot using a His•Tag monoclonal antibody from mouse (Novagen) and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Invitrogen, Molecular Probes) as the secondary antibody. His•Tag-ChvI purification using nickel-affinity chromatography was performed in the laboratory of Professor Bi-Cheng Wang at University of Georgia (USA).
Electrophoretic mobility shift assay using genomic DNA (GD.EMSA)
To prepare samples, S. meliloti Rm1021 genomic DNA was digested to completion by overnight incubation with Bsp143I restriction enzyme (Sau3AI isoschizomer, Fermentas Life Sciences, Canada) and the reaction was then heat-inactivated. Purified His•Tag-ChvI protein was mixed with digested DNA in a solution of 9% glycerol, 3 mM acetyl phosphate, 0.8 mM Tris-acetate, 0.25 mM magnesium acetate, 1.65 mM potassium acetate, 2.5 μg ml-1 bovine serum albumin (BSA). For negative controls, ChvI protein was not added to samples. Incubations were carried out for 30 minutes at room temperature and loaded directly on gel without dye.
To perform the electrophoresis, a sodium boric acid buffer (SB buffer) was made following the specifications of Brody and Kern [52]. 5% nondenaturing polyacrylamide gels (14 cm × 16 cm) were cast using a Hoefer SE 600 gel electrophoresis unit and following the standard procedure for resolution of small DNA fragments [53] but using SB buffer instead of TBE buffer. Gels were run in 1X SB buffer between 25 to 40 mA for 3–6 hours. Gels were then stained for 1 hour in a 3X GelRed™ staining solution containing 0.1 M NaCl and following manufacturer's recommendation for post gel staining (Biotium, USA, CA) prior to visualization on a UV transilluminator. Shifted DNA bands in the highest part of the gel were then excised and stored in 2-ml plastic tubes at −20°C.
To recover DNA fragments from polyacrylamide gel, the method from Ausubel et al. (1992) [53] was used. The elution buffer used contained 0.5 M ammonium acetate, 1 mM EDTA, 0.1% SDS and final pH 8. 200 μl elution buffer was added to each tube containing a piece of gel. The gel was then crushed in smaller pieces using a pipet tip. Tubes were incubated overnight at 37°C with shaking. Following centrifugation in a microcentrifuge at room temperature for 10 minutes at 10,000 rpm, supernatant was removed and transferred to a clean 2.0 ml tube. Ethanol (500 μl) was added to precipitate the DNA and tubes were placed at −20°C overnight. DNA was pelleted at 13,000 rpm for 10 minutes. Supernatant was removed and DNA solubilized in 100 μl of 10 mM Tris pH 8 and 15 μl of 5 M sodium chloride was added. DNA was then precipitated a second time with 2 volumes of ethanol and kept overnight at −20°C. Precipitated DNA was recovered by centrifugation in a microcentrifuge at 13,000 rpm for 15 minutes, supernatant was removed and DNA was dried. Final resuspension of DNA was done with 10 μl of 10 mM Tris pH 8.
The DNA fragments were cloned into the BamHI site in pUC18. Prior to ligation, BamHI-digested pUC18 was dephosphorylated using shrimp alkaline phosphatase (Fermentas Inc.) and the reaction stopped by heat-inactivation. Ligation was performed overnight at room temperature with T4 DNA ligase (Fermentas Inc.). Transformation of calcium chloride competent E. coli DH5α cells was done following standard procedure [54]. Over 40 transformant colonies were streak-purified from each experiment. A selection of them were then used for plasmid preparation and tested for the presence of an insert using restriction digest with EcoRI and PstI. Fragments cloned in pUC18 were sequenced using primers M13F provided by the sequencing facility (University of Waterloo) or LB61 (Table 3).
Sequences were first analyzed by searching for Sau3AI (Bsp143I) restriction sites to determine the limits of each fragment. Each fragment sequence was then searched against S. meliloti Rm1021 genomic sequence using the BLAST tool from Toulouse annotation website [55]. Genes in closest proximity to identified sequences and potentially regulated by ChvI were searched against STRING 8.1 databases (June 28, 2009) for functional relations [23]. The search was directed from the Toulouse annotation website.
Reporter gene fusion strains
Transcriptional fusion strains were obtained by transduction from the reporter gene fusion library strains made by Cowie et al. [20]. SmFL strains were used to prepare transduction lysates to transfer the gene fusions from the original S. meliloti RmP110 background into the Rm1021 background. Selection of transductants was done on LB with gentamicin (60 μg ml-1). The same lysates were also used to transduce gene fusions into SmUW38 (pKD001) with selection on LB gentamicin (60 μg ml-1) and neomycin (200 μg ml-1). Four transductants per transduction experiment were picked and streaked on LB gentamicin and neomycin. Transductants were then cured of pKD001 by streaking them on MM9-succinate gentamicin (20 μg ml-1) containing 2.5% sucrose and incubated at 30°C for four days. pDK001-cured strains were finally streaked on MM9-succinate gentamicin.
Phage ΦM12 was used for transductions following the usual procedure [56], except that TY media was used instead of LBmc media to prepare and dilute lysates. High yield of transductants required the use of Bacto™-Agar, -Tryptone, and -Yeast extract (BD). Diluted lysate (0.5 ml) was mixed with equal volume of cell suspension and incubated at room temperature for 30 minutes. Cells were then recovered by centrifugation in a microcentrifuge for 10 minutes and washed twice with 2 ml of saline. Final resuspension was done with 400 μl saline and then spread on two agar plates. Plates were incubated at 30°C for four days.
Growth in liquid media
Inocula were prepared by resuspending bacterial biomass from MM9-succinate-agar plates into a saline solution (0.85% NaCl) to obtain an optical density (OD600) of 0.8. Test tubes containing 5-ml liquid media made of MM9-succinate with/without 0.1% proline and/or 0.1% uracil where inoculated with the inoculum at a 10% concentration. Test tubes were incubated at 30°C with constant shaking. Growth was monitored by reading the absorbance at 600 nm. Growth rate constants (μ) were calculated based on absorbance values during the exponential growth phase and using the formula: μ = ( (log10 N - log10 N0) 2.303) / (t - t0). Results represent the average of duplicates and the standard deviation was calculated as the error.
β-Glucuronidase assay
To measure transcription from reporter gene fusion strains, the β-glucuronidase assay described in Cowie et al. [20] was adapted. Strains were grown in MM9-succinate plus 0.1% proline, 0.1% uracil, and gentamicin until OD600 of 0.2 - 0.8. These cells were then used directly for the assay in microplates as described previously [20]. Assays were done in triplicate and standard deviation calculated.
Competing interests
The authors declare that they have no competing interest.
Authors' contributions
LB planned and carried out experiments, performed data analysis, and wrote the manuscript. TCC planned experiments and wrote the manuscript. Both authors read and approved the final manuscript.
This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to T.C.C. L.B. received a scholarship from "Fonds québécois de la recherche sur la nature et les technologies" (FQRNT).
We thank Professor Bi-Cheng Wang and Dr. Hao Xu at University of Georgia (USA) for provision of the purified ChvI protein and Professor Turlough M. Finan from McMaster University (Canada) who made the fusion library available to us. We are grateful to Jennifer Moore and Jacquelyn Fleming for technical assistance, Dr. Jiujun Cheng for critically reading the manuscript, and Kathy Lam and John Heil for assistance with data analysis.
Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer ELL, Eddy SR, Bateman A: The Pfam protein families database.
Nucleic Acids Res 2010, 38:D211-D222. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Galperin MY: Structural classification of bacterial response regulators: diversity of output domains and domain combinations.
J Bacteriol 2006, 188:4169-4182. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gao R, Stock AM: Biological insights from structures of two-component proteins.
Annu Rev Microbiol 2009, 63:133-154. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Charles TC, Nester EW: A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens.
J Bacteriol 1993, 175:6614-6625. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sola-Landa A, Pizarro-Cerdá J, Grilló MJ, Moreno E, Moriyón I, Blasco JM, Gorvel JP, López-Goñi I: A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence.
Mol Microbiol 1998, 29:125-138. PubMed Abstract | Publisher Full Text
Viadas C, Rodríguez MC, Sangari FJ, Gorvel JP, García-Lobo JM, López-Goñi I: Transcriptome analysis of the Brucella abortus BvrR/BvrS two-component regulatory system.
PLoS One 2010, 5:e10216. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Quebatte M, Dehio M, Tropel D, Basler A, Toller I, Raddatz G, Engel P, Huser S, Schein H, Lindroos HL, Andersson SGE, Dehio C: The BatR/BatS two-component regulatory system controls the adaptive response of Bartonella henselae during human endothelial cell infection.
J Bacteriol 2010, 192:3352-3367. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Vanderlinde EM, Yost CK: Mutation of the sensor kinase chvG in Rhizobium leguminosarum negatively impacts cellular metabolism, outer membrane stability, and symbiosis.
J Bacteriol 2012, 194:768-777. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Cheng HP, Walker GC: Succinoglycan production by Rhizobium meliloti is regulated through the ExoS-ChvI two-component regulatory system.
J Bacteriol 1998, 180:20-26. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bélanger L, Dimmick KA, Fleming JS, Charles TC: Null mutations in Sinorhizobium meliloti exoS and chvI demonstrate the importance of this two-component regulatory system for symbiosis.
Mol Microbiol 2009, 74:1223-1237. PubMed Abstract | Publisher Full Text
Osterås M, Stanley J, Finan TM: Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.
J Bacteriol 1995, 177:5485-5494. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wang C, Kemp J, Da Fonseca IO, Equi RC, Sheng X, Charles TC, Sobral BWS: Sinorhizobium meliloti 1021 loss-of-function deletion mutation in chvI and its phenotypic characteristics.
Mol Plant Microbe Interact 2010, 23:153-160. PubMed Abstract | Publisher Full Text
Chen EJ, Sabio EA, Long SR: The periplasmic regulator ExoR inhibits ExoS/ChvI two-component signalling in Sinorhizobium meliloti.
Mol Microbiol 2008, 69:1290-1303. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lu H-Y, Luo L, Yang M-H, Cheng H-P: Sinorhizobium meliloti ExoR is the target of periplasmic proteolysis.
J Bacteriol 2012, 194:4029-4040. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Pinedo CA, Gage DJ: HPrK regulates succinate-mediated catabolite repression in the gram-negative symbiont Sinorhizobium meliloti.
J Bacteriol 2009, 191:298-309. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wells DH, Chen EJ, Fisher RF, Long SR: ExoR is genetically coupled to the ExoS-ChvI two-component system and located in the periplasm of Sinorhizobium meliloti.
Mol Microbiol 2007, 64:647-664. PubMed Abstract | Publisher Full Text
Chen E, Fisher R, Perovich V, Sabio E, Long S: Identification of direct transcriptional target genes of ExoS/ChvI two-component signaling in Sinorhizobium meliloti.
J Bacteriol 2009, 191:6833-6842. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Garner MM, Revzin A: A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.
Nucleic Acids Res 1981, 9:3047-3060. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Liu P, Wood D, Nester EW: Phosphoenolpyruvate carboxykinase is an acid-induced, chromosomally encoded virulence factor in Agrobacterium tumefaciens.
J Bacteriol 2005, 187:6039-6045. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Cowie A, Cheng J, Sibley CD, Fong Y, Zaheer R, Patten CL, Morton RM, Golding GB, Finan TM: An integrated approach to functional genomics: construction of a novel reporter gene fusion library for Sinorhizobium meliloti.
Appl Environ Microbiol 2006, 72:7156-7167. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Caspi R, Altman T, Dreher K, Fulcher CA, Subhraveti P, Keseler IM, Kothari A, Krummenacker M, Latendresse M, Mueller LA, Ong Q, Paley S, Pujar A, Shearer AG, Travers M, Weerasinghe D, Zhang P, Karp PD: The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases.
Nucleic Acids Res 2012, 40:D742-D753. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y: KEGG for linking genomes to life and the environment.
Nucleic Acids Res 2008, 36:D480-D484. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J, Doerks T, Julien P, Roth A, Simonovic M, Bork P, von Mering C: STRING 8--a global view on proteins and their functional interactions in 630 organisms.
Nucleic Acids Res 2009, 37:D412-D416. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Arias A, Cerveñansky C: Galactose metabolism in Rhizobium meliloti L5-30.
J Bacteriol 1986, 167:1092-1094. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Geddes BA, Oresnik IJ: Inability to catabolize galactose leads to increased ability to compete for nodule occupancy in Sinorhizobium meliloti.
J Bacteriol 2012, 194:5044-5053. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
López-Lara IM, Sohlenkamp C, Geiger O: Membrane lipids in plant-associated bacteria: their biosyntheses and possible functions.
Mol Plant Microbe Interact 2003, 16:567-579. PubMed Abstract | Publisher Full Text
Vences-Guzmán MA, Geiger O, Sohlenkamp C: Sinorhizobium meliloti mutants deficient in phosphatidylserine decarboxylase accumulate phosphatidylserine and are strongly affected during symbiosis with alfalfa.
J Bacteriol 2008, 190:6846-6856. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Neural Network Promoter Prediction.
[http://www.fruitfly.org/seq_tools/promoter.html webcite]
Barton LL, Johnson GV, Schitoskey K, Wertz M: Siderophore-mediated iron metabolism in growth and nitrogen fixation by alfalfa nodulated with Rhizobium meliloti.
J Plant Nutr 1996, 19:1201-1210. Publisher Full Text
O Cuív P, Clarke P, Lynch D, O'connell M: Identification of rhtX and fptX, novel genes encoding proteins that show homology and function in the utilization of the siderophores rhizobactin 1021 by Sinorhizobium meliloti and pyochelin by Pseudomonas aeruginosa, respectively.
J Bacteriol 2004, 186:2996-3005. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lynch D, O'Brien J, Welch T, Clarke P, Cuív PO, Crosa JH, O'Connell M: Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti.
J Bacteriol 2001, 183:2576-2585. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Viguier C, O Cuív P, Clarke P, O'connell M: RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011.
FEMS Microbiol Lett 2005, 246:235-242. PubMed Abstract | Publisher Full Text
Chao T-C, Buhrmester J, Hansmeier N, Puhler A, Weidner S: Role of the regulatory gene rirA in the transcriptional response of Sinorhizobium meliloti to iron limitation.
Appl Environ Microbiol 2005, 71:5969. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Beck S, Marlow VL, Woodall K, Doerrler WT, James EK, Ferguson GP: The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis.
Microbiology (Reading, Engl) 2008, 154:1258-1270. Publisher Full Text
Griffitts JS, Long SR: A symbiotic mutant of Sinorhizobium meliloti reveals a novel genetic pathway involving succinoglycan biosynthetic functions.
Mol Microbiol 2008, 67:1292-1306. PubMed Abstract | Publisher Full Text
Jacob AI, Adham SAI, Capstick DS, Clark SRD, Spence T, Charles TC: Mutational analysis of the Sinorhizobium meliloti short-chain dehydrogenase/reductase family reveals substantial contribution to symbiosis and catabolic diversity.
Mol Plant Microbe Interact 2008, 21:979-987. PubMed Abstract | Publisher Full Text
Mauchline TH, Fowler JE, East AK, Sartor AL, Zaheer R, Hosie AHF, Poole PS, Finan TM: Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome.
Proc Natl Acad Sci USA 2006, 103:17933-17938. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Chen H, Teplitski M, Robinson JB, Rolfe BG, Bauer WD: Proteomic analysis of wild-type Sinorhizobium meliloti responses to N-acyl homoserine lactone quorum-sensing signals and the transition to stationary phase.
J Bacteriol 2003, 185:5029-5036. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Clover RH, Kieber J, Signer ER: Lipopolysaccharide mutants of Rhizobium meliloti are not defective in symbiosis.
J Bacteriol 1989, 171:3961-3967. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Djordjevic SP, Ridge RW, Chen HC, Redmond JW, Batley M, Rolfe BG: Induction of pathogenic-like responses in the legume Macroptilium atropurpureum by a transposon-induced mutant of the fast-growing, broad-host-range Rhizobium strain NGR234.
J Bacteriol 1988, 170:1848-1857. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Newman JD, Diebold RJ, Schultz BW, Noel KD: Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.
J Bacteriol 1994, 176:3286-3294. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Noel KD, Diebold RJ, Cava JR, Brink BA: Rhizobial purine and pyrimidine auxotrophs: Nutrient supplementation, genetic analysis, and the symbiotic requirement for the novo purine biosynthesis.
Arch Microbiol 1988, 149:499-506. Publisher Full Text
Danielli A, Roncarati D, Delany I, Chiarini V, Rappuoli R, Scarlato V: In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis.
J Bacteriol 2006, 188:4654-4662. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Foreman DL, Vanderlinde EM, Bay DC, Yost CK: Characterization of a gene family of outer membrane proteins (ropB) in Rhizobium leguminosarum bv. viciae VF39SM and the role of the sensor kinase ChvG in their regulation.
J Bacteriol 2010, 192:975-983. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Dozot M, Poncet S, Nicolas C, Copin R, Bouraoui H, Mazé A, Deutscher J, De Bolle X, Letesson J-J: Functional characterization of the incomplete phosphotransferase system (PTS) of the intracellular pathogen Brucella melitensis.
PLoS One 2010, 5:e12679. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM: Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis.
J Bacteriol 1982, 149:114-122. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Galibert F, Finan T, Long S, Pühler A, Abola P, Ampe F, Barloy-Hubler F, BARNETT M, Becker A, Boistard P, Bothe G, Boutry M, Bowser L, Buhrmester J, Cadieu E, Capela D, Chain P, Cowie A, Davis R, Dreano S, Federspiel N, FISHER R, Gloux S, Godrie T, Goffeau A, Golding B, Gouzy J, Gurjal M, Hernández-Lucas I, Hong A, et al.: The composite genome of the legume symbiont Sinorhizobium meliloti.
Science 2001, 293:668-672. PubMed Abstract | Publisher Full Text
Hanahan D: Studies on transformation of Escherichia coli with plasmids.
J Mol Biol 1983, 166:557-580. PubMed Abstract | Publisher Full Text
Studier FW, Moffatt BA: Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
J Mol Biol 1986, 189:113-130. PubMed Abstract | Publisher Full Text
Yanisch-Perron C, Vieira J, Messing J: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene 1985, 33:103-119. PubMed Abstract | Publisher Full Text
Kaniga K, Delor I, Cornelis GR: A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica.
Gene 1991, 109:137-141. PubMed Abstract | Publisher Full Text
Brody JR, Kern SE: History and principles of conductive media for standard DNA electrophoresis.
Anal Biochem 2004, 333:1-13. PubMed Abstract | Publisher Full Text
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Short protocols in molecular biology. 2nd edition. New York: Greene Publishing Associates and John Wiley and Sons; 1992.
Sambrook J, Russell DW: Molecular cloning: a laboratory manual, Vol 1-3. 3rd edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2001.
Sinorhizobium meliloti 1021.
[http://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi webcite]
Finan TM, Hartweig E, Lemieux K, Bergman K, Walker GC, Signer ER: General transduction in Rhizobium meliloti.
J Bacteriol 1984, 159:120-124. PubMed Abstract | Publisher Full Text | PubMed Central Full Text


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5 Oct
Rare 'Sofa Shark' Surprises Scientists, Who Promptly Let Him Go
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A rare deepwater species has been spotted by scientists in Scotland for the first time in over a decade, offering the world a fresh reminder of what fascinating forms lurk beneath the ocean's depths.
Rare "sofa shark" found off Hebrides http://t.co/c8NWxTyi3D pic.twitter.com/uytR552ora
— Scotsman (@TheScotsman) September 30, 2015
The bizarre-looking fish, known as both a "sofa shark" or "false catshark," was captured by a team of marine biologists from the Scottish Shark Tagging Programme. Weighing in at over 130 pounds, with a length of 6 feet, the rare shark's appearance left scientists stunned.
"I was pretty surprised when it landed in our boat," marine biologist Francis Neat told The Scotsman. "We quickly measured and weighed it before sending it back into the water. We hadn't seen one in ten years."
Read more about the discovery on The Scotsman.
Remember when Jeremy Wade caught a rare Glyphis shark? Relive the moment here!


text: Adoptables Sale!Soo, I just wanted to sell all the old adoptables I have left.I'll maybe start making new ones when they are sold. If you want to buy one, just note me.:iconthe-cookiezz: is my adoptables accountIf you buy 2 adoptables you get a third one for half the price. #3 is open #2 and #3 are open the winged fox is soldyou can find all these adoptables here!


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Education: Academic freedomEditorial: Government plans will leave teachers asking what exactly free schools have to do with professional freedom
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The children's rape trial shows we need an urgent review of the system
Response: We have to ask how a child will be affected by cross-examination in an adult court, says Maggie Atkinson
Save this beacon of philosophical studyLetters: The closing of Middlesex University's philosophy department would be disastrous for the academic and intellectual life of this country


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This property allows you to book online with a credit card. After submitting a reservation request, the owner or manager has 24 hours to accept, at which time your card will be charged and you will receive an email confirmation
Recent Mesquite reviews
"My foursome has been coming to Mesquite for several years now, staying at the local motel/casinos. This year we tried something new and we're never going back! This place is waaaay more comfortable! Well stocked kitchen, BBQs, pool. After a day out on the golf course, we really appreciated the hot tube and the giant recliner sofas. The owners were super easy to work with, too! Looking forward to next year!"Guest: Craig J. Date of Stay: April 2015 Review Submitted: May 4, 2015View listing
"This condo was excellent for our golf trip. It slept 6 comfortably and we had plenty of room. Very clean with easy access to pool and hot tub. Would definitely stay here again."Guest: Ben Date of Stay: February 2015 Review Submitted: March 12, 2015View listing
"We were two couples meeting in Mesquite. We found that everything in the previous reviews was true. We were concerned about the house being located near an airport, but we only heard one plane during our 5-night stay, and it did not bother us at all. The floor plan is excellent for two couples. The bedrooms and bathrooms are completely separated, so we never disturbed each other. We appreciated the fact that it is all on one level with no stairs. We liked the house's location, central to the areas we were there to explore: Little Finland and Valley of Fire in Nevada, and Snow Canyon in Utah. The restaurant at the Wolf Creek golf course is convenient, walking distance away. However, during our stay it closed at 6 p.m. on weekdays. We agree that the house looks even better in person than the photos."Guest: SA Date of Stay: April 2015 Review Submitted: April 17, 2015View listing
"My family was ecstatic about their accomodations. Nuff Said. Grandpa"Guest: Pops Date of Stay: November 2014 Review Submitted: December 3, 2014View listing
Featured Destinations: Big Bear Cabin Rentals, San Diego Vacation Rentals, Lake Tahoe Cabin Rentals, Black Butte Ranch


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Mehdi Hasan exposes misconceptions about the continent
Explore Japan through its offbeat train culture
25 May 2013 14:45 GMT | Sport, Formula One, Germany, Monaco, Spain
Germany's Nico Rosberg scorched to his third successive Formula One pole position with team mate Lewis Hamilton completing a front row lockout for Mercedes at the Monaco Grand Prix on Saturday.The pole was the fourth in a row for Mercedes, whose qualifying form has faded in the races so far this season, andsecond successive sweep of the top two places.Red Bull's triple world champion Sebastian Vettel qualified third, on a drying track after a damp session, with Australian team mate and last year's winner Mark Webber lining up alongside.Title contender Kimi Raikkonen, four points behind Vettel after five races, starts fifth for Lotus with Ferrari's FernandoAlonso in sixth place for a race where overtaking can be extremely tricky.Rosberg could become the first son of a Monaco Grand Prix winner to win the most glamorous race on the calendar - 30 years on from his world champion father Keke's 1983 victory for Williams - but he was not about to get ahead of himself."It's always nice to hear statistics like that but it doesn't change much for tomorrow," said the Monaco resident who grew up in the Mediterranean principality and had dominated every practice session through the treacherous metal-fenced streets. "In Barcelona we were struggling a lot in the race and I finished 70 seconds behind the winner. We're improving step by step, but there are still a lot of questions remaining. It (the tyre wear) is still our weakness but we'll make the best of it and who knows."
'Massive improvement'
Hamilton, 0.91 slower than his team mate, was happy to overcome the problems he experienced in practice and to qualify second for a race that should see just two pitstops rather than the average of four witnessed in Spain."I've been struggling all weekend and my pace has been slacking. I've struggled with the car and this is the first timethat's happened since 2005," the 2008 winner said, referring to his pre-F1 days.
"It's been a difficult weekend so I'm happy to have been able to lock out the front row for the team. It's been a massive improvement for me."
"I've been struggling all weekend and my pace has been slacking. I've struggled with the car and this is the first time that's happened since 2005 "
- Mercedes driver Lewis Hamilton
A light shower before qualifying threw an element of uncertainty into the mix, with Venezuelan Pastor Maldonado fastest for Williams at the end of the first phase on a drying track that caught out Force India's Paul Di Resta.Ferrari's Felipe Massa did not set a time after crashing in final practice, leaving his mechanics with too much to fix and not enough time to do it. The Brazilian will start last.Frenchman Romain Grosjean was also racing against time after also hitting the barriers in the morning but the Lotus driver managed to get out of the pits and into the second phase before complaining that he had been held up by a Toro Rosso.With Massa and Di Resta out, Caterham's Dutch rookie Giedo van der Garde made it into the second phase for the first time.
Vettel was quickest in the second phase but could not match the Mercedes when it mattered most."It was very close. Mercedes were very quick all weekend. We know they're quick over one lap but I don't want that to sound like an excuse," said the German.
"If anyone is to blame, then it's me. The car was very good."
Source: Reuters
Mercedes to start the Spanish Grand Prix from the front as Nico Rosberg and Lewis Hamilton finish fastest in qualifying.
Sport, Formula One
Mercedes take their second consecutive pole at the Bahrain GP as teammate Lewis Hamilton picks up a five-place penalty.
Sport, Formula One, Bahrain, China, Germany
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Precarious peace agreement has failed to end fighting in South Sudan with fears of famine and disease outbreaks.
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While targeting 'terrorists and junkies', Kremlin-backed strongman Kadyrov flaunts the law to safeguard moderate Islam.
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101 East follows the lives of seven people in Nanjing and asks what they think of the direction their country is taking.
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Hello, I am super excited to be home tutoring and virtual teaching again. I have been working as a teacher in various educational environments for the last 17 years. I am currently working for UCLA Health as a Instructional Technologist Triage Support Help Desk Agent while attending graduate school at California State Stanislaus in Turlock and pursuing my master's degree in school administration. I have tremendous skills when it comes to working as a certificated teacher. I use a positive classroom management system to control classroom behavior, keep students on task and teach with high expectations. I use a teaching resource kit that provides professional curriculum in grades K-8 and strategies for working with students of all abilities. I am professional in my manner, dress, and adore working with children and all staff in the educational arena. Upon accepting a teaching/tutoring assignment, I immediately lesson plan from my kit those materials necessary to use in case lesson plans are not available. I use many up to date student engagement strategies and differentiated instruction when necessary. I am fluent with techniques that maintain a high rate of positive teacher to student interactions and create risk free student response opportunities. I maintain high expectation for all students and set learning objectives in a clear and concise manner. I use methods such as TAPPLE to check for student understanding "real time" and re-teach if necessary. I love teaching.
I believe I am an exemplary candidate for teaching/tutoring teaching for because I am well educated, highly certificated, and absolutely love working with students. I am currently working on my thesis (perceptions from teachers' who were coached by peer coaches) for my master's degree in education with an emphasis in school administration. I am also pursuing my reading certificate so that some day I can work with semi-illiterate and illiterate youth and adults. I have a multiple subject CLAD credential, Advanced SDAIE Certificate, and my NCLB in multiple subjects. Teaching is my passion and I believe I was born to be a teacher. It is in my blood—I am constantly enhancing my skills as a teacher/tutor through working as a certificated trainer, volunteering as a reading intervention teacher, and athletic coaching. I believe some of my innate abilities are working with children. I am creative and constantly add a twist or two to make my lessons more interesting. I also have tremendous active listening skills coupled a multitude of questioning techniques. My favorite part of teaching is developing positive relationships with my students. I can see when students are struggling and instinctively know when a quiet word of encouragement is needed. Students know I understand and care about them.
I have many outside experiences that add to my teaching/tutoring talents. I have traveled all over the world working as an adventure tour guide and white water river guide. I worked for 11 seasons as a commercial Grand Canyon River Guide. Additionally, I have traveled to various other continents to play women's water polo for the US women's national team and compete in swimming at NCAA division III championships. I believe in talking well planned, research based, smart risks and it has always paid off. Lastly, I have a positive attitude towards life and towards teaching/tutoring to inspire students to do their best. I believe that by setting realistic goals I am able to maintain my positive and realistic attitude. Hello, I am super excited to be home tutoring and virtual teaching again. I have been working as a teacher in various educational environments for the last 17 years. I am currently working for UCLA Health as a Instructional Technologist Triage Support Help Desk Agent while attending graduate school at California State Stanislaus in Turlock and
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I am currently a masters competitive swimmer who has many times in the top ten in the national for my age group. I have been teaching swimming for over 30 years and coaching swimming for over 25 years. I have been head swim coach for Calaveras High School Boys and Girls for 5 years. In 1993, I played for the US womens water polo team in Brazil. I have also coached water polo for various youth camps.
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Vagabond Inn Sunnyvale
Today:Open 24 Hours
816 W. Ahwanee Ave.,
Sunnyvale, CA 94085
(408) 734-4607
Hotel Rooms
Secure online booking with Expedia Affliate Network Call 866-761-3335 to book by phone
The room rates listed are double occupancy per room unless otherwise stated and exclude tax recovery charges and service fees.
Business Details
YP Qualified
|Great Value. Friendly People.
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Regular Hours
- General Info:
Property Location With a stay at Vagabond Inn Sunnyvale in Sunnyvale, you'll be close to Cogswell Polytechnical College and Computer History Museum. This motel is within close proximity of Mountain View Center for the Performing Arts and California's Great America.
Rooms Make yourself at home in one of the 60 air-conditioned guestrooms. Complimentary wireless Internet access keeps you connected, and cable programming is available for your entertainment. Bathrooms have complimentary toiletries and hair dryers. Conveniences include safes and desks, as well as phones with free local calls.
Rec, Spa, Premium Amenities Take advantage of recreation opportunities such as an outdoor pool, or other amenities including complimentary wireless Internet access and complimentary use of a nearby fitness facility.
Dining Enjoy a satisfying meal at a restaurant serving guests of Vagabond Inn Sunnyvale. A complimentary breakfast is included.
Business, Other Amenities Featured amenities include complimentary high-speed (wired) Internet access, complimentary newspapers in the lobby, and a 24-hour front desk. Free parking is available onsite.
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Fax: (408) 734-1675
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- English, Spanish
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Robert F. Gates LEBANON Robert (Bob) Franklin Gates, 94, died at Cornwall Manor on Sunday, January 12, 2014. Born in Durant, Okla., on August 31, 1919, to Wallace Wesley and Dora Albright Gates, he was the third of six children. Bob was drafted into the
during WWll while he was at Oklahoma A&M College, he enlisted in the Army Air Corps as a pilot. Upon completion of pilot training he was commissioned a second lieutenant and assigned to the Ferry Command delivering all types of aircraft throughout the US and overseas. He received the Air Medal for the first successful delivery of P-38 aircraft over the North Atlantic. Following this service he was assigned as Captain in the Air Transport Command flying C-54 transports from Wilmington, Delaware to the border of China. It was while stationed at New Castle, Del., that he met Jean Barr Howell, and married her on May 1, 1943. While in the service, he became interested in working to improve aviation safety, so upon discharge he moved his family to Kansas City, Mo., in 1945 and joined the Civil Aeronautics Administration as a Flight Operations Inspector assigned to supervise TWA. In 1949 he became Section Chief in Washington, D.C. for low visibility runway operations, which later moved to the FAA Technical Center near Atlantic City, NJ. During this time the family resided in Ocean City, NJ. His work continued for 20 years until his retirement from the FAA in 1972 when he received the prestigious FAA Achievement Award. In retirement he organized a consulting business, Gates Associates, which kept him busy advising on airport lighting systems for many domestic and international airports. After six years of consulting, Bob and Jean fully retired to Buck Hill Falls in the Poconos, where they enjoyed golf, tennis, lawn bowling and cross country skiing. After 20 years in Buck Hill they moved to Cornwall Manor. Bob was also a popular Explorer Scout Master and Sunday School teacher for many years. He sang in church choirs for over 75 years and also held several lay positions in various churches He was a devoted husband, and loving father having encouraged growth in arts and careers. He leaves behind Jean Howell Gates, 94 at the Health Center of Cornwall Manor; son, Henry (Jaqueline) of Cambridge, Mass; daughters, Virginia (Frank) May of Buck Hill Falls, and Elizabeth (Richard) Petrunis of Mays Landing, N.J.; eight grandchildren Sandra Cassel, John May, Joshua Gates, Jessica Makovsky, Erica Gates, Kristopher Gates, Evan Petrunis and John Petrunis; and five great-grandchildren, Mason and Gina Cassel, Makalya and Mila Gates and Ashley Makovsky; and two sisters, Della Craighead and Lois Lenz. He was predeceased by his eldest son, Robert Jr (Molly) a prominent documentary film maker from Charleston, W.Va. He was also predeceased by sisters, Rubie Bryant and Dora Mae Yates; and brother, Walter Gates. A memorial service will be held at 11 a.m. Thursday, January 16, 2014 at the Cornwall United Methodist Church, Cornwall Pike & Alden Sts., Cornwall, with Rev. James Heath officiating. The family will receive visitors following the service. Interment will be on a later date at Fort Indiantown Gap National Cemetery, Annville. The family requests in lieu of flowers that a contribution in the name of Robert Gates be made to the
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By Catholic Online (NEWS CONSORTIUM)
5/12/2012 (3 years ago)
Catholic Online (www.catholic.org)
The question arises - can a horror movie be too scary? The latest anthology horror film, "V/H/S," about a group of teenagers finding a box of cursed videotapes in a haunted house poses that question. When the film screened at the Sundance Film Festival earlier this year, a man and woman fled the auditorium to vomit - but it seems there was a different factor behind the couple's dinner "return engagement."
By Catholic Online (NEWS CONSORTIUM)
Catholic Online (www.catholic.org)
5/12/2012 (3 years ago)
Published in Movies
Keywords: Dolby, horror movies, technological advances, V/H/S
LOS ANGELES, CA (Catholic Online) - During a graphic and gory scene, the man ran from the theater, collapsed in the lobby and had a seizure. His girlfriend followed suit and vomited while he was being treated. An ambulance was called.
The man and his girlfriend were fine after being treated by paramedics.
It seems the incident had little to with the gory images, but rather from the film's sound. Horror films are about to get even more nauseating thanks to the latest technology from Dolby Laboratories.
"When someone is hiding in a basement and there are footsteps on the floorboards above, the system will actually play that audio from above in the theater," Stuart Bowling of Dolby Laboratories says.
Dolby has revealed its new sound system, the Dolby Atmos, capable of pumping out sound how we naturally hear it: from every direction. Unlike current sound systems that push sound from the sides of theaters, the Atmos system can not only deliver sound from above, it will be able to swirl sound around the theater.
Bowling says that the new Atmos system is one of the biggest technological advances the company has made in the last 20 years.
"You can imagine watching a scary movie, and it's a scene when someone is hiding in a basement and there are footsteps on the floorboards above," Bowling said, according to the New York Times. "The Atmos system will actually play that audio from above people in the theater."
The new system has 64 speaker feeds and will be in select theaters later this year.
"Dolby Atmos is our most significant innovation in years and represents the future for entertainment sound in cinema," President and Chief Executive Officer, Dolby Laboratories, Kevin Yeaman said in a press release.
"We have leveraged our deep insights into how people hear and experience sound to empower filmmakers, studios, and exhibitors with new technology that transforms storytelling."
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By Greg Goodsell (NEWS CONSORTIUM)
Director Steven Spielberg returns to the screen with "Bridge of Spies," an incisive look into Cold War-era America. His first feature film since "Lincoln," Spielberg focuses on the real-life trade off a Soviet Spy in exchange for an American service man downed ... continue reading
By Greg Goodsell (NEWS CONSORTIUM)
"The Martian," a space opera about astronaut Matt Damon being stranded on the Red Planet, is doing astronomical business at the nation's box offices. In the manner of "Gravity" in 2013, an outer space suspense yarn that took moviegoers by storm, many analysts ... continue reading
By Atarah Haely (NEWS CONSORTIUM)
Randall Wallace, known for his films Braveheart, Heaven is For Real, Pearl Harbor and The Man in the Iron Mask, said he was unsuccessful in life until he asked God to help him.After he asked God for guidance, Wallace went from jobless to becoming one of Hollywood's ... continue reading
By Nikky Andres (NEWS CONSORTIUM)
A new documentary has been created centered around abortion doctor Kermit Gosnell, titled 3801 Lancaster: American Tragedy. The trailer is set to be released Monday night. MUNTINLUPA, PHILIPPINES (Catholic Online) - The feature-length documentary has been in production ... continue reading
By Nikky Andres (NEWS CONSORTIUM)
Film writer and director Peter Spencer has announced his attempt to raise awareness about the critical situation of Christians being persecuted in the Middle East through his film Return to the Hiding Place. It is about a group of people who fight to save the lives of ... continue reading
By Catholic Online (NEWS CONSORTIUM)
A quiet domestic drama about a family who turns to prayer -- "War Room" opened at theaters nationwide this past weekend. "War Room" spoke to an unspoken need among moviegoers, as its box office receipts far overtook the debuting action drama "No Escape" and the ... continue reading
By Greg Goodsell (NEWS CONSORTIUM)
As a director of horror films, Wes Craven definitely had his vicissitudes. He had some disappointments, and not all of his films spun gold at the box office. He would remain world-famous had he only directed three of his films, "Last House on the Left" in 1972, ... continue reading
By Catholic Online (NEWS CONSORTIUM)
Rabid Scientology adherent and movie star Tom Cruise is building a $50 million dollar state-of-the art studio on five acres in Hollywood. Word is out that the station will broadcast news with a Scientological slant - "A Scientology CNN," and spread the ... continue reading
By Atarah Haely (NEWS CONSORTIUM)
During a recent Church Boys podcast, David A.R. White shared some details about the upcoming sequel to the 2014 Christian movie hit "God's Not Dead." The actor-producer and co-founder of the production company, Pure Flix Entertainment explained that the follow-up movie ... continue reading
By Catholic Online (NEWS CONSORTIUM)
It's a sign of the times... Fabled action star Sylvester Stallone will take his Rambo character in an all-out assault against ISIS in Syria and Iraq in the final film in the series, "Last Blood." LOS ANGELES, CA (Catholic Online) - The fifth installment in the ... continue reading


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By this time, most readers of Lab Soft News will be familiar with the rapidly expanding Volkswagen scandal whereby the company has knowingly modified the software installed its on-board car computers for years. Emission controls on its vehicles were being activated only when they were being tested and deactivated then when the cars were being driven on the road (see: Volkswagen: The scandal explained). In this way, the cars were able to meet regulatory emission requirements but also achieve better fuel mileage on the road while contaminating the air with nitrous oxides, imperiling the health of many people.
Car manufacturers like Volkswagen regard their software, consisting of millions of lines of code, as proprietary so this massive fraud has escaped detection for years.The company is finally being brought to justice by the EPA which enforces much stricter emission guidelines than the environmental regulatory bodies in Europe and Germany. The latter have a cozy relationship with Volkswagen and the boundaries between company and German government are fuzzy at best. This scandal will ultimately cost Volkswagen many tens of billions of dollars and irreparable harm to its reputation and is emblematic of corporate greed.
Now consider the parallels with the EHR companies whose products are critical in the management of most hospital and office patients in the country. Such software, also consisting of millions of lines of code, is viewed as proprietary by the EHR companies and, as such, is protected from close scrutiny. Recent evidence has emerged that these EHR companies include "gag clauses" in their contracts with hospitals with severe penalties if hospital personnel discuss their EHRs in public (Gag Clauses in EHR Contracts Documented; Concerns Raised about Patient Safety). Given that all of the software, manuals, and even screen shots are viewed as proprietary, this amounts to a total prohibition of public discussion of EHR problems. Such discussions are confined solely to the vendors themselves. Recent blog notes have raised the topic of the "Epic police" who enforce these contractual gag orders. Here's an example from HIStalk about a problem this past April at HIMSS (see: From HIMSS 4/16/15):
...[a HIStalk reporter] reported an encounter with the "Epic police" this week. Epic was demonstrating in the Interoperability Showcase and...[she]...snapped a photo of what Epic was publicly displaying. Someone from Epic came up, said nobody's allowed to take pictures of Epic's screens, and then demanded that the photo be deleted from the camera while they stood there to verify. That sounds like Soviet-style overstepping big time, especially since...[she] was attending as a journalist – if you are showing your product on the big screen, if HIMSS doesn't prohibit picture-taking in the exhibit hall.... and if the person taking the photo doesn't work for an organization that has signed a confidentiality agreement with Epic, then I don't believe Epic people have any legal basis for detaining attendees and demanding that they delete photos.
Here's more information about the "Epic police" from the KevinMD blog by Dr. David Mann (see: It's time to remove EHR gag orders. For patient safety's sake):
A couple of years ago I got beaten up by Epic and my hospital administration for publishing a tongue-in-cheek satirical review of the Epic EHR as a video game, complete with screenshots. Epic didn't like the screenshots and ordered them removed. As it turns out, Epic has a squad of workers (some have referred to them as the "Epic Police") whose sole job is to scour the Internet, looking for Epic screenshots, and then bullying the poster until they are removed. The whole sordid tale is told here. I am not the only one who has had this experience. Several of my colleagues have had run-ins with the Epic Police, with similar results.
I am sure that a sizable number of patients are harmed on a yearly basis by EHR software. Such injuries are usually not the result of software bugs alone but rather faulty interactions between physicians/nurses and the EHR software. For the harmed patients, however, this distinction provides little solace. Because of contractual gag clauses, such errors will usually be discussed and managed by hospital personnel and/or reported to the EHR vendors who will theoretically make the necessary software changes across their client base to avoid future errors. We need a bette resolution of problems such as these. I am opposed to rigid regulatory control over EHR software. The current inadequate EHR products would probably become even more fossilized with strict FDA oversight. However, just imagine the long-term consequences of the Volkswagen malfeasance if the EPA had not stepped in. None of this skulduggery might ever have come to light. We urgently need open and public discussions of the various problems associated with EHR "proprietary" software (see, for example: Reducing EHR "Wrong Orders" by Limiting the Number of "Open Charts"; NYT Op-Ed on EHR Interoperability Blames Vendors and Greedy Hospitals).


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The economy is getting better? I quit!
Sally Herships
Oct 31, 2013 A report out today shows more people are quitting their jobs.